Project Summary Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by elevated pressure in the pulmonary arteries. Pulmonary arterial hypertension (WHO Group I PH; PAH) is a particularly severe form of PH frequently associated with right heart failure and premature death. There is no cure, and treatments only target the symptoms. Approximately 50% of PAH patients die within 5 years of diagnosis. There is therefore a compelling, unmet need for new therapeutic strategies. Pulmonary vascular remodeling is the defining pathological feature of PAH. It leads to occlusion of distal pulmonary arterioles, with accompanying increase in pulmonary vascular resistance. Vascular remodeling is promoted by the survival and proliferation of pulmonary arterial vascular cells under conditions of oxidative stress and in the presence of DNA damage. Here we provide evidence that the Eyes Absent protein (EYA3) promotes survival of DNA damaged cells facing a survival-versus-apoptosis decision. EYA3 is a druggable and mechanistically unique protein tyrosine phosphatase (PTP) present at elevated levels in pulmonary arterial smooth muscle cells isolated from PAH patients. In proof of principle studies, we show that transgenic mice harboring an inactivating mutation in the EYA3 PTP domain are significantly protected from vascular remodeling in a chronic hypoxia model, and that inhibitors of EYA3 PTP activity reverse vascular remodeling in a rat model of experimental angio-obliterative PH. The overall goal of this project is to establish the EYA3-PTP as a disease-modifying target whose function in the pathophysiology of PAH can be targeted by available inhibitors. This will be a critical milestone in pre-clinical drug target validation and will be achieved through the use of genetic and pharmacological approaches in the following two Specific Aims: Aim I: To elucidate the molecular mechanism(s) through which EYA3 promotes the pathogenesis of PAH we will use a murine chronic hypoxia model. Hemodynamic and histopathological analyses upon cell-type specifc EYA3 deletion will be conducted to identify the cell-autuonomous roles of EYA3 in vascular remodeling. Single-cell transcriptomics and phospho-protein analyses will be used to delineate the molecular mechanims contributing to vascular remodeling. Aim II: To establish the effectiveness of EYA3-PTP inhibitors in reversing established vascular remodeling we will use a rat model of angio-obliterative PAH and evaluate the most safe and effective modes of EYA3-targeted pharmacological intervention. The overall impact of this proposal is that it will define a targetable signaling mechanism that contributes to the characteristic pulmonary vascular pathobiology in PAH, demonstrate that EYA3-PTP inhibitors are viable lead compounds for the development of PAH therapeutics, and provide insights into a previously unexplored molecular mechanism contributing to PAH pathology.